Highly diluted and well-mixed yellow-green fluorescent microspheres
(FluoSpheres, 1.0 mm diameter; Invitrogen, Molecular
Probes; F8823) were added as an internal reference to
all samples. Readings were collected in logarithmic mode (at
least 5000 events per sample) and analysed with FlowJo
v10.0.7r2 (FlowJo LLC, Oregon). Data was collected using GFL/
side scatter (SSC) dot plots and specified gates taken from
Brussaard et al. (2010), V1, V2 and V3 which correspond to
viruses of differing fluorescence intensity (total
count ¼ V1þV2þV3). This enabled optimal distinction between
stained viruses and other microbial cells and/or background
noise, thus filtration to remove such particles wasn't
required. Blanks, consisting of TE-buffer and autoclaved 0.2-
mm-filtered sample, were pre-treated and analysed identically
to samples, further facilitating the correction of virus counts
for noise.